We continue to develop and apply Atomic Force Microscopy (AFM) to basic and applied biophysical studies of malaria. In addition, we are nearing completion of a new and advanced form of Near-Field Scanning Optical Microscope (NSOM) that will allow us to elucidate nanoscopic features below the diffraction limit of light. NSOM can correlate topographic to fluorescence data through their concurrent acquisition.. Like our AFMs, our NSOM will be the first instrument of its kind deployed at the NIH for biomedical research. We are using these scanning probe instruments in two malaria-related projects. For example, it has been shown that individuals with Hemoglobin C (HbC) have a survival advantage against fatal malaria in that it does not protect against infection but can protect against severe malaria. Preliminary transmission electron microscopy (TEM) data indicated that malaria-infected, homozygous HbCC cells may have abnormally large or aggregated surface knobs compared with normal hemoglobin HbAA-infected cells. However, a basic failing of the TEM for this type of research is that it is limited to visualization of only the contour of the cell membrane. By AFM, as we have previously shown, we can provide detailed quantitative analyses of the topography of the cell and correlate it using associated fluorescence microscopy with the developmental stage of the parasite inside the cell. Our early data indicate that there are marked quantitative differences in size and distribution of knobs between HbCC and HbAA cells. The unusually large knob complexes may be related to the immunological protective effect(s) of CC cells against severe malaria. For example, aggregated knobs and protein band 3 could make the malaria-infected cells more susceptible to IgG as has been shown in other vertebrate cell aging systems.). Our work many clarify these aspects of the HbCC protection mechanism. The physical-chemical properties of lipid membranes are crucially important to many biological processes such as the invasion of infectious agents, sub-cellular trafficking and the application of therapeutic agents. We have expanded upon our unique study of the thermodynamic transition enthalpy, entropy and number of lipids in a typical domain of 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), using three component lipid systems containing cholesterol, a critical component of biological membranes. We have succeeded in developing AFM-based analytical methods that can reveal modifications in nanoscopic domains resulting from the insertion of cholesterol. We have used our AFM data to generate phase diagrams with unique and previous unknown critical points that provide new insights into physical chemical processes in supported biological membrane systems. This work is under review in the Biophysical Journal. In collaboration with the National Institute of Standard and Technology (NIST), have also obtained unique nanometer scale domain formations with mixed lipid samples using two complimentary fluorescent lipid dyes and a custom-made NSOM. We succeeded in following the distribution of lipid molecules as they formed membrane domains. The work was presented at The 7th International Conference on Near-field Optics. The next phase of this project involves the use of actual biological membranes. as part of our plan to analyze and characterize increasingly complex membrane systems. We are now studying mammalian rhodopsin-containing native disk membranes as a prelude to our goal of studying erythrocyte membranes and their modification during the course of a malaria infection. The rhodopsin membrane system was chosen because of the wealth of physical-chemical data available. Our research is critical to a better overall understanding of membrane ?rafts,? in general, and is being carried out in collaboration with the National Institute of Alcohol Abuse and Alcoholism (NIAAA). Our work using scanning probe microscopy technology is being supplemented with classical cell biological, molecular biological, and physical chemical studies of changes in proteins, most specifically flotillins and band 3, associated with erythrocyte membranes during the malaria intra-erythrocytic cycle. Work is in progress on the sporozoite stage of a mosquito infection using GFP-labeled sporozoites to elucidate the nature of sporozoite attractant(s) demonstrated by this laboratory to exist. Analyses of biophysical studies of changes in erythrocyte ?flicker? resulting from a malaria infection are completed and the work is being prepared for publication. A new project to determine the influence of erythrocyte age on its infectability by Plasmodium falciparum has been initiated.